Human AB Research Articles

Expanding CAR T cells in human platelet lysate renders T cells with in vivo longevity

BackgroundPre-clinical and clinical studies have shown that the infusion of CAR T cells with a naive-like (TN) and central memory (TCM) phenotype is associated with prolonged in vivo T cell persistence and superior anti-tumor effects. To optimize the maintenance of such populations during the in vitro preparation process, we explored the impact of T cell exposure to both traditional [fetal bovine serum (FBS), human AB serum (ABS)] and non-traditional [human platelet lysate (HPL) - a xeno-free protein supplement primarily used for the production of clinical grade mesenchymal stromal / stem cells (MSCs)] serum supplements.MethodsSecond generation chimeric antigen receptor with CD28 and CD3ζ endodomain targeting prostate stem cell antigen (PSCA) (P28z) or CD19 (1928z) were constructed and used for this study. After retroviral transduction, CAR T cells were divided into 3 conditions containing either FBS, ABS or HPL and expanded for 7 days. To evaluate the effect of different sera on CAR T cell function, we performed a series of in vitro and in vivo experiments.ResultsHPL-exposed CAR T cells exhibited the less differentiated T cell phenotype and gene signature, which displayed inferior short-term killing abilities (compared to their FBS- or ABS-cultured counterparts) but superior proliferative and anti-tumor effects in long-term in vitro coculture experiments. Importantly, in mouse xenograft model, HPL-exposed CAR T cells outperformed their ABS or FBS counterparts against both subcutaneous tumor (P28z T cells against Capan-1PSCA) and systemic tumor (1928z T cells against NALM6). We further observed maintenance of less differentiated T cell phenotype in HPL-exposed 1928z T cells generated from patient’s PBMCs with superior anti-tumor effect in long-term in vitro coculture experiments.ConclusionsOur study highlights the importance of serum choice in the generation of CAR T cells for clinical use.

Safety and Anti-Tumor Activity Study of Loncastuximab Tesirine and Durvalumab in Diffuse Large B-Cell, Mantle Cell, or Follicular Lymphoma

Introduction: Loncastuximab tesirine (Lonca) is an antibody-drug conjugate (ADC) comprising a humanized anti-CD19 antibody (Ab) conjugated to a pyrrolobenzodiazepine dimer toxin. In a Phase 1, first-in-human ADCT-402-101 clinical study, Lonca demonstrated single-agent anti-tumor activity with manageable toxicity in patients with relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL) and follicular lymphoma (FL). Durvalumab is a human monoclonal Ab of the immunoglobulin G-1 kappa subclass that blocks the interaction of programmed death-ligand 1 (PD-L1) with PD-1 on T-cells and with CD80 (B7.1) on other immune cells. Blockade of PD-L1/PD-1 and PD-L1/CD80 interactions releases the inhibition of immune responses, including those that may result in tumor elimination. Preclinical data, as well as early results from clinical trials combining ADCs and checkpoint inhibitors, show potentially increased effectiveness of these therapeutics when used in combination and provide the rationale for the current trial. Study Design and Methods: This is a Phase 1b, open-label, dose escalation (Part 1) and expansion (Part 2) trial of Lonca combined with durvalumab in patients with R/R DLBCL, MCL, or FL (NCT03685344). The key inclusion and exclusion criteria for the ADCT-402-104 study are reported in Table 1, and the dosing schema is presented in Figure 1. This trial will evaluate the safety and tolerability, preliminary anti-tumor activity, pharmacokinetics, pharmacodynamics, and immunogenicity of Lonca combined with durvalumab. Patients will receive Lonca once every 3 weeks for 2 doses in total, and durvalumab every 4 weeks for up to 1 year. Patients with only partial response or stable disease at the second disease evaluation may receive 2 additional doses of Lonca given once every 3 weeks. During Part 1, the dose of Lonca will be escalated using a classic 3+3 design with a fixed dose of durvalumab. Part 2 will consist of up to 3 expansion cohorts, one for each of the DLBCL, MCL, and FL populations. All patients in Part 2 will receive the dose of Lonca determined in Part 1, with a fixed dose of durvalumab. The trial opened in February 2019 and recruitment is ongoing. Study sponsored by ADC Therapeutics SA with the support of MedImmune Limited, a wholly-owned subsidiary of AstraZeneca Pharmaceuticals PLC, which supplies durvalumab (http://clinicaltrials.gov/show/NCT03685344). Disclosures Moskowitz: ADC Therapeutics: Research Funding; Merck: Consultancy, Research Funding; Celgene: Consultancy; Pharmacyclics: Research Funding; Seattle Genetics, Inc.: Consultancy, Research Funding; Genentech: Consultancy, Research Funding. Ungar:ADC Therapeutics: Employment, Other: Stock options. equity interest. Dautaj:ADC Therapeutics: Employment, Other: Stock options.

A Human IgE Antibody Binding Site on Der p 2 for the Design of a Recombinant Allergen for Immunotherapy.

Der p 2 is one of the most important allergens from the house dust mite Dermatophagoides pteronyssinus Identification of human IgE Ab binding epitopes can be used for rational design of allergens with reduced IgE reactivity for therapy. Antigenic analysis of Der p 2 was performed by site-directed mutagenesis based on the x-ray crystal structure of the allergen in complex with a Fab from the murine IgG mAb 7A1 that binds an epitope overlapping with human IgE binding sites. Conformational changes upon Ab binding were confirmed by nuclear magnetic resonance using a 7A1-single-chain variable fragment. In addition, a human IgE Ab construct that interferes with mAb 7A1 binding was isolated from a combinatorial phage-display library constructed from a mite-allergic patient and expressed as two recombinant forms (single-chain Fab in Pichia pastoris and Fab in Escherichia coli). These two IgE Ab constructs and the mAb 7A1 failed to recognize two Der p 2 epitope double mutants designed to abolish the allergen-Ab interaction while preserving the fold necessary to bind Abs at other sites of the allergen surface. A 10-100-fold reduction in binding of IgE from allergic subjects to the mutants additionally showed that the residues mutated were involved in IgE Ab binding. In summary, mutagenesis of a Der p 2 epitope defined by x-ray crystallography revealed an IgE Ab binding site that will be considered for the design of hypoallergens for immunotherapy.

Effects of human serum on the stability and cytotoxicity of silver nanoparticles

Silver nanoparticles are ubiquitous in today’s commercial marketplace. Despite decades of research, there is still disagreement about the risk that silver nanoparticles pose to human and environmental health. There is often a lack of correlation between measured physical properties and the measured biological endpoints, making it difficult to establish trends, and discrepancies between different cell models have not been clearly accounted for. Here we have used human AB serum in cell culture media for the investigation in place of fetal bovine serum (FBS) which is typically used. In HepG2 cells, particle uptake was clearly correlated with smaller sizes of particles and with increased cytotoxicity. In neuron-like SH-SY5Y cells, similar trends were not observed. Compared with previously published data on the same particles in these cell lines with cell culture media supplemented with FBS, there are clear and significant differences in the stability of the particles, their cellular uptake and their cytotoxicity in these two commonly used cell models for in vitro cytotoxicity testing. This highlights the need for improved in vitro testing for nanomaterials and the importance of using human serum to better predict human in vivo outcomes.

Novel human Ab against vascular endothelial growth factor receptor 2 shows therapeutic potential for leukemia and prostate cancer.

Vascular endothelial growth factor receptor 2 (VEGFR2) is highly expressed in tumor‐associated endothelial cells, where it modulates tumor‐promoting angiogenesis, and it is also found on the surface of tumor cells. Currently, there are no Ab therapeutics targeting VEGFR2 approved for the treatment of prostate cancer or leukemia. Therefore, development of novel efficacious anti‐VEGFR2 Abs will benefit cancer patients. We used the Institute of Cellular and Organismic Biology human Ab library and affinity maturation to develop a fully human Ab, anti‐VEGFR2‐AF, which shows excellent VEGFR2 binding activity. Anti‐VEGFR2‐AF bound Ig‐like domain 3 of VEGFR2 extracellular region to disrupt the interaction between VEGF‐A and VEGFR2, neutralizing downstream signaling of the receptor. Moreover, anti‐VEGFR2‐AF inhibited capillary structure formation and exerted Ab‐dependent cell‐mediated cytotoxicity and complement‐dependent cytotoxicity in vitro. We found that VEGFR2 is expressed in PC‐3 human prostate cancer cell line and associated with malignancy and metastasis of human prostate cancer. In a PC‐3 xenograft mouse model, treatment with anti‐VEGFR2‐AF repressed tumor growth and angiogenesis as effectively and safely as US FDA‐approved anti‐VEGFR2 therapeutic, ramucirumab. We also report for the first time that addition of anti‐VEGFR2 Ab can enhance the efficacy of docetaxel in the treatment of a prostate cancer mouse model. In HL‐60 human leukemia‐xenografted mice, anti‐VEGFR2‐AF showed better efficacy than ramucirumab with prolonged survival and reduced metastasis of leukemia cells to ovaries and lymph nodes. Our findings suggest that anti‐VEGFR2‐AF has strong potential as a cancer therapy that could directly target VEGFR2‐expressing tumor cells in addition to its anti‐angiogenic action.

1490PD - A phase Ia/IIa trial of Sym015, a MET antibody mixture, in patients with advanced solid tumours

BackgroundMET gene amplification/mutation is implicated in oncogenesis of many tumor types. Sym015 is a recombinant antibody (Ab) mixture containing 2 humanized Abs binding non-overlapping epitopes on MET which has superior preclinical activity compared to other MET Abs. Here we present interim results from the First in Human study of Sym015. MethodsIn part 1 dose escalation, Sym015 was evaluated on every 2-week (q2w) schedule at 6/12/18/24mg/kg doses in late line advanced solid tumor patients (pts). Based on PK/PD assessment, 18mg/kg loading dose followed by 12mg/kg q2w was selected as RP2D. The on-going Part 2 Dose expansion enrolls pts with MET amplification (METAmp >5 MET copies by Next Generation Sequencing (NGS) or FISH MET/CEP7 ratio >2.2 updated to≥3.0) and/or MET exon 14 deletion (METEx14Δ) positive tumors. Central FISH confirmation of tumor MET status and longitudinal circulating tumor (ct)DNA profiling is performed. ResultsBy April 2019, 51 pts (median age 61 yrs) have been treated, 12 pts in Part 1 and 39 pts in Part 2, the majority are non small cell lung cancer (NSCLC; 14 pts; 6 METAmp,7 METEx14Δ, 1 both) and gastric cancer (GC; 12 METAmp pts). Treatment-related adverse events (TRAE) occurred overall in 41,2%; TRAE ≥ gr3 in 9,8% (edema, colitis, septic shock, hypoalbuminemia, hypophosphatemia, increased amylase). No pts discontinued or died due to TRAE. The most common TRAE of≥5% were fatigue 15.7%, peripheral edema 7,8%, nausea, decreased appetite, pruritus and abdominal pain reported by 5.9% of pts each. The table shows preliminary efficacy in NSCLC patients. Sym015PK was slightly non-linear, with a dose-dependent t½ of 7-10 days for the first dose. 83% METAmp concordance (10/12 samples) between tumor and blood was observed.Table1490PDTableNSCLC PtMET Status (Local Assessment)Best Response (Best % Change From Baseline)Previous MET TKI therapyDuration of Response (weeks)1METAmp NGS 6.8 CopiesPR (-53%)No802METAmp NGS >5 CopiesSD (-29%)No9, Ongoing3METAmp NGS >5 CopiesSD (+3%)No9, Ongoing4METEx14ΔSD (-28%)No7, Ongoing5METAmp FISH MET/ CEP7:4.9SD (+6%)No116METEx14ΔSD (-4%)Yes87METEx14ΔSD (-21%)Yes158METEx14ΔSD (-20%)Yes89METAmp SISH MET/ CEP7: 15.2PendingNoOngoing10METEx14ΔPendingYesOngoing11METEx14Δ + METAmpPendingPendingOngoing12METEx14ΔPendingNoOngoing13METEx14ΔPendingNoOngoing14METEx14ΔNot EvaluableYesNot Evaluable ConclusionsSym015 was well tolerated with early evidence of antitumor activity. Predictive biomarker analysis is underway. The study continues to enroll NSCLC patients with METAmp and/or METEx14Δ. MET screening in blood seems feasible and may be included in future trials. Clinical trial identificationNCT02648724. Legal entity responsible for the studySymphogen A/S. FundingSymphogen A/S. DisclosureD.R. Camidge: Advisory / Consultancy: 2019: Takeda, CBT Pharmaceuticals, Daiichi-Sankyo (ILD adjudication committee), G1 Therapeutics (DSMB), Bio-Thera (DSMB), Blueprint; Advisory / Consultancy: 2018: AstraZeneca, Takeda, Arrys/Kyn, Regeneron, Hengrui, G1 Therapeutics (DSMB), Daiichi Sankyo (ILD adjudication committee), Hansoh (SRC), Bio-Thera (DSMB), Ribon, BMS, Blueprint, Roche/Genentech, Inivata; Advisory / Consultancy: 2017: Genoptix, G1 Therapeutics (DSMB), Mersana Therapeutics, Roche/Genentech, Takeda, Ignyta, Daiichi Sankyo (ILD adjudication committee), Hansoh SRC, Bio-Thera DSMB, Lycera, Revolution Med; Research grant / Funding (self), Research grant / Funding (institution): 2017: Takeda Investigator-initiated Trial; Research grant / Funding (self), Research grant / Funding (institution), Company Sponsored Trials at Institution: 2018/9: AbbVie, AstraZeneca, BMS, Hansoh, Lycera, MedImmune, Merck, Pfizer, Phosplatin, Roche/Genentech, Seattle Genetics, Symphogen, Takeda. F. Janku: Research grant / Funding (self): Novartis, Genentech, BioMed Valley Discoveries, Astellas, Agios, Plexxikon, Deciphera, Piqur, Symphogen, Bristol-Myers Squibb, Asana, Upsher-Smith Laboratories; Advisory / Consultancy: Guardant Health, IFM Therapeutics, Synlogic, Deciphera; Advisory / Consultancy, Shareholder / Stockholder / Stock options: Trovagene; Advisory / Consultancy: Immunomet. D.V. Catenacci: Honoraria (self), Advisory / Consultancy: Merck, BMS, Lilly, Astellas, Gritstone, Taiho, Five Prime, Genentech Roche, Foundation Medicine, Guardant Health, Tempus. H.C. Chung: Honoraria (self): Merck-Serono, Lilly, Foundation Medicine; Advisory / Consultancy: Taiho, Celltrion, MSD, Lilly,Quintiles, BMS, Merck-Serono; Research grant / Funding (institution): Lilly, GSK, MSD, Merck-Serono, BMS-ONO, Taiho. A. Dowlati: Advisory / Consultancy: Seattle genetics, Takeda, AbbVie; Research grant / Funding (institution): Takeda, Taiho, Roche, EMD Serono, Bayer, Tesaro, Regeneron, Amgen, Mirati, BMS. K.S. Rohrberg: Research grant / Funding (institution), Travel / Accommodation / Expenses: Roche; Research grant / Funding (institution): Loxo Oncology, Orion Pharma, Pfizer, PUMA, Cantargia, Genmab, Novartis, Incyte, Bayer, AstraZeneca, Alligator, Merck, BMS; Travel / Accommodation / Expenses: Sanofi; Research grant / Funding (institution): Symphogen. T.T. Poulsen: Full / Part-time employment: Symphogen A/S. H. Rudbæk: Full / Part-time employment: Symphogen A/S. L. Alifrangis: Full / Part-time employment: Symphogen A/S. R.P. Dalal: Full / Part-time employment: Symphogen A/S. All other authors have declared no conflicts of interest.

Assessing Propylene Glycol Interference at Concentrations Relevant to Serum Levels With Electronic Cigarette Use in an Enzymatic Ethylene Glycol Assay

Abstract Introduction Clinical laboratory measurement of ethylene glycol (ETG) by enzymatic methods is known to be subject to interference with high (several hundred milligrams/deciliter) concentrations of propylene glycol (PG). An estimated 10.8 million people in the United States, or almost 1 in 20 Americans, are reported to use electronic cigarettes (e-cigarettes; ie, “vaping”), in which PG is frequently used as a liquid solvent. No published reports exist investigating whether serum concentrations of PG that may be seen with e-cigarette use (generally, concentrations of 10-15 mg/dL in moderate users and up to 30 mg/dL in heavy users) can lead to either interference flags in analytical ETG measurement or falsely positive ethylene glycol concentrations being reported. Methods Studies were conducted to assess PG interference on enzymatic ETG measurement. Commercially acquired human AB serum (catalog # 35-060-CI; Corning Life Sciences) was spiked with various amounts of PG (catalog #158720010; Acros Organics/Thermo Fisher Scientific) to achieve both concentrations of PG expected with e-cigarette use (0-30 mg/dL) and supra-physiological concentrations (>30 mg/dL) for comparative purposes. PG-spiked sera were tested using a quantitative enzymatic ETG assay (Quantitative Ethylene Glycol kit, catalog #C504-0A; Catachem) previously validated as a laboratory-developed test on an AU5810 automated chemistry analyzer (Beckman Coulter). Results None of the tested PG concentrations designed to mimic physiological levels expected with e-cigarette use (0, 5, 10, 15, 20, 25, and 30 mg/dL) caused falsely positive quantitative ETG results, defined as values above the ETG limit of quantitation (LOQ) of 5 mg/dL. At these low concentrations, interference patterns neither were observed in reaction monitors, nor were instrument interference error flags triggered. As concentrations of PG approached 50 mg/dL, abnormal curve appearances were observed in the instrument reaction monitor, although this neither resulted in interference error flags nor false detection of ETG. However, as PG concentrations approached and exceeded ≥100 mg/dL, both interference flags and falsely positive ETG results were observed. Conclusion Results from the present experiments demonstrate that serum concentrations of PG expected with e-cigarette use did not cause interference with enzymatic ethylene glycol detection in this assay.

Human Platelet Lysate Media Supplement Supports Lentiviral Transduction and Expansion of Human T Lymphocytes While Maintaining Memory Phenotype.

Immune cell therapy has emerged as a promising approach to treat malignancies that were up until recently only treated on a palliative basis. Chimeric antigen receptor- (CAR-) modified T lymphocytes (T cells) in particular have proven to be very effective for certain hematological malignancies. The production of CAR T cells usually involves viral transduction and ex vivo culture of T cells. The aim of this study was to explore the use of human platelet lysate (HPL) compared to two commonly used supplements, human AB serum (ABS) and fetal bovine serum (FBS), for modified T cell production. For studying transduction, activated T cells were transduced with lentivirus to deliver GFP transgenes with three different promoters. Transduction efficiency (percent GFP) was similar among the supplements, and a modest increase in the transgene product (mean fluorescence intensity) was observed when HPL was used as a supplement compared to ABS. To study the effect of supplements on expansion, peripheral blood mononuclear cells (PBMCs) were activated and expanded in the presence of interleukin 2 (IL2) for fourteen days. T cell expansions using HPL and ABS were comparable and slightly less than the expansion obtained with FBS. Interestingly, cells expanded in media supplemented with HPL showed a higher percentage of T cells with a central memory phenotype compared to those expanded in ABS or FBS. Protein profiling revealed that the phenotypic differences may be explained by elevated levels of several cytokines in HPL, including IL7. The results suggest that the use of HPL as a cell culture supplement during the production of modified T cells is a reasonable alternative to ABS. Furthermore, the use of HPL may enhance in vivo performance of the final product by enriching for central memory T cells that are associated with long-term persistence following adoptive transfer.

Characterization of the human myelin oligodendrocyte glycoprotein antibody response in demyelination

Over recent years, human autoantibodies targeting myelin oligodendrocyte glycoprotein (MOG Ab) have been associated with monophasic and relapsing central nervous system demyelination involving the optic nerves, spinal cord, and brain. While the clinical relevance of MOG Ab detection is becoming increasingly clear as therapeutic and prognostic differences from multiple sclerosis are acknowledged, an in-depth characterization of human MOG Ab is required to answer key challenges in patient diagnosis, treatment, and prognosis. Herein, we investigated the epitope, binding sensitivity, and affinity of MOG Ab in a cohort of 139 and 148 MOG antibody-seropositive children and adults (n = 287 patients at baseline, 130 longitudinal samples, and 22 cerebrospinal fluid samples). MOG extracellular domain was also immobilized to determine the affinity of MOG Ab. MOG Ab response was of immunoglobulin G1 isotype, and was of peripheral rather than intrathecal origin. High affinity MOG Ab were detected in 15% paediatric and 18% adult sera. More than 75% of paediatric and adult MOG Ab targeted a dominant extracellular antigenic region around Proline42. MOG Ab titers fluctuated over the progression of disease, but affinity and reactivity to Proline42 remained stable. Adults with a relapsing course intrinsically presented with a reduced immunoreactivity to Proline42 and had a more diverse MOG Ab response, a feature that may be harnessed for predicting relapse. Higher titers of MOG Ab were observed in more severe phenotypes and during active disease, supporting the pathogenic role of MOG Ab. Loss of MOG Ab seropositivity was observed upon conformational changes to MOG, and this greatly impacted the sensitivity of the detection of relapsing disorders, largely considered as more severe. Careful consideration of the binding characteristics of autoantigens should be taken into account when detecting disease-relevant autoantibodies.

Displacement Affinity Release of Antibodies from Injectable Hydrogels.

Current methods to tune release rates of therapeutic antibodies (Abs) for local delivery are complex and routinely require bioconjugations that may reduce Ab bioactivity. To rapidly tune release profiles of bioactive Abs, we developed a biophysical interaction system within a neutravidin modified poly(carboxybetaine) hydrogel (pCB-NT) that tunes release rates of desthiobiotinylated Abs (D-Abs) using a constant hydrogel and D-Ab combination. Herein, we delivered desthiobiotinylated bevacizumab (D-Bv), a recombinant humanized monoclonal IgG1 Ab for antiangiogenic cancer therapies. D-Bv's high affinity for pCB-NT (KD 7.8 × 10-10 M; t1/2 ∼ 2 h) produces a slow D-Bv release rate (∼5 ng day-1) that is increased by the dissolution of hydrogel encapsulated biotin derivative pellets, which displaces D-Bv from pCB-NT binding sites. In contrast to traditional affinity systems, displacement affinity release of Abs (DARA) does not require Ab or hydrogel modifications for each unique release rate. D-Bv release rates were tuned by simply altering the total biotin derivative concentration; the effective first-order (keff) and mass per day release rates were tuned 25- and 8-fold, respectively. Local surface plasmon resonance (LSPR) and biolayer interferometry (BLI) confirmed the D-Bv binding affinity for the corresponding ligand and Fc receptor, demonstrating that the biophysical interaction system is amenable to anticancer Abs for receptor or cytokine blockade and immune cell recruitment to cancer cells.

Non-pooled Human Platelet Lysate: A Potential Serum Alternative for In Vitro Cell Culture

Serum supplementation is crucial in in vitro cell culture to provide all the essential nutrients needed for cellular processes. Fetal bovine serum (FBS) is considered the 'gold standard', but its production raises serious ethical concerns. Human-derived alternatives to FBS exist in the form of human platelet lysates (hPLs) or human AB serum (ABS). However, these serum products are usually pooled from several donors, in order to have a standardised product without patient-specific deviations. Nevertheless, the use of patient-specific serum in cell culture might be the key to successful transplantation of the cultured cells in medical applications, particularly as it avoids the transmission of infectious components or xenogenic proteins. In addition, the production of non-pooled hPL from single donors is likely to be a cost-effective and time-saving method. The current study used hPL units isolated from single donors and tested their performance as medium supplements for cell culture in comparison with FBS or ABS. This proof-of-concept study aimed to assess the potential of non-pooled hPL for personalised serum supplementation, and thus optimise in vitro models by making them more relevant to human physiology. We showed that A549, HepG2 and Caco-2 human cell lines were generally able to adapt to the new culture conditions and maintain viability, morphology and certain cell-specific characteristics. These results indicate that non-pooled, single patient-derived hPL could be a suitable alternative for in vitro serum supplementation.

Characterization of the Angiogenic Potential of Human Regulatory Macrophages (Mreg) after Ischemia/Reperfusion Injury In Vitro.

Ischemia/reperfusion- (I/R-) induced organ damage represents one of the main causes of death worldwide, and new strategies to reduce I/R injury are urgently needed. We have shown that programmable cells of monocytic origin (PCMO) respond to I/R with the release of angiogenic mediators and that transplantation of PCMO results in increased neovascularization. Human regulatory macrophages (Mreg), which are also of monocytic origin, have been successfully employed in clinical transplantation studies due to their immunomodulatory properties. Here, we investigated whether Mreg also possess angiogenic potential in vitro and could represent a treatment option for I/R-associated illnesses. Mreg were differentiated using peripheral blood monocytes from different donors (N = 14) by incubation with M-CSF and human AB serum and stimulation with INF-gamma. Mreg cultures were subjected to 3 h of hypoxia and 24 h of reoxygenation (resembling I/R) or the respective nonischemic control. Cellular resilience, expression of pluripotency markers, secretion of angiogenic proteins, and influence on endothelial tube formation as a surrogate marker for angiogenesis were investigated. Mreg showed resilience against I/R that did not lead to increased cell damage. Mreg express DHRS9 as well as IDO and display a moderate to low expression pattern of several pluripotency genes (e.g., NANOG, OCT-4, and SOX2). I/R resulted in an upregulation of IDO (p < 0.001) while C-MYC and KLF4 were downregulated (p < 0.001 and p < 0.05). Proteome profiling revealed the secretion of numerous angiogenic proteins by Mreg of which several were strongly upregulated by I/R (e.g., MIP-1alpha, 19.9-fold; GM-CSF, 19.2-fold; PTX3, 5.8-fold; IL-1β, 5.2-fold; and MCP-1, 4.7-fold). The angiogenic potential of supernatants from Mreg subjected to I/R remains inconclusive. While Mreg supernatants from 3 donors induced tube formation, 2 supernatants were not effective. We suggest that Mreg may prove beneficial as a cell therapy-based treatment option for I/R-associated illnesses. However, donor characteristics seem to crucially influence the effectiveness of Mreg treatment.

250-LB: Affinity Purification and Identification of Unique, Shared, Anti-IA-2 B-Cell Clonotypes: A Proteomic Approach to Understanding Pathophysiological Mechanisms Underpinning Humoral Autoimmunity in Type 1 Diabetes

Immune destruction of beta (β) cells is a feature of type 1 diabetes (T1D), yet the pathophysiological role of autoantibodies (Abs) is poorly understood. Recently, proteomic interrogation of Abs indicates that restricted heavy (H) and light (L) chain gene usage underpins targeting of classic autoantigens in systemic autoimmunity. In the current study, we have taken a similar approach to characterise B cell clonotypes resulting in targeting of the major T1D autoantigen, IA-2. Firstly, we developed a novel ELISA assay for the major IA-2 epitopes using an MBP-fusion protein (FP) incorporating intracellular domain of IA-2. We next established ELISA-based affinity purification (AP) method using the IA-2-MBP FP to isolate enriched IA-2 specific Abs from 2 patients with verified (by RSR commercial ELISA) anti-IA-2 autoimmunity. The AP IA-2 Abs were then analysed for Ig H and L chain clonality, variable region gene usage and AA sequence using high-res electrophoretic fractionation and de-novo/database-driven analysis of AA peptides (TripleTOF 5600+ MS). We report the presence of 2 unique Ig Kappa-restricted clonotypes giving rise to IA-2 autoimmunity; namely a IGHV3.23 H chain paired with either a IGKV1.9 or IGKV3.20 L chain. Remarkably, both clones were present in AP Ig from 2 unrelated T1D patients, with shared AA replacement mutations. The Ig signatures were not present in healthy, age/sex matched controls, T1D patients negative for anti-IA-2, or Lupus. These extraordinary findings suggest the pathogenic mechanisms leading to IA-2 autoimmunity may be virtually identical across individuals, leading to the intriguing possibility of intervention in the processes underpinning the disease. Furthermore, the ability to AP and identify genuine human clonal IA-2 Abs will unlock functional studies aimed at establishing the role of these Abs in the pathophysiology of β cell loss. Disclosure T. Mendis: None. J. Wang: None. M. Pietropaolo: None. M.W. Jackson: None.

Evaluation of L-index interference limits on Roche cobas c502 and c702 immunoturbidimetric assays using endogenously lipemic specimens and intralipid spiking

ObjectivesSpecimen lipemia is a primary concern with turbidimetric and nephelometric assays due the potential interference caused by light scattering or absorption. The purpose of this study was to evaluate lipemic interference thresholds across seven FDA-cleared assays using patient specimens with varying degrees of endogenous lipemia pre- and post-ultracentrifugation (UC) and with Intralipid spiking. MethodsUsing an IRB-approved protocol, residual human serum specimens (n=42; L-indices, 1-1769; H-index ≤85; I-index ≤2) were obtained. Baseline and post-UC testing was conducted across assays on cobas c502 and c702 instruments (Roche Diagnostics; Indianapolis, IN). Serum indices and triglyceride (TRIG) concentrations were also measured pre- and post-UC. Intralipid spiking studies with human AB serum were also conducted. Lipoprotein subfraction analysis (Lipoprint; Quantimetrix; Redondo Beach, CA) was performed on three additional patient specimens with elevated TRIG post-UC to determine which TRIG-containing lipoprotein fraction(s) remain. ResultsSeveral assays showed L-index thresholds derived from endogenously lipemic specimens that were below previously defined limits from the package inserts (PIs) [new (prior)]: AAT 400 (500); CERU 100 (200); HAPTO 450 (600); TRSF 250 (500). L-index limits derived from Intralipid spiking were generally higher than those listed in PIs. UC did not adversely impact results in non-lipemic or lipemic specimens. UC was effective at clearing lipemic interference, although persistence of residual VLDL was often observed. ConclusionsThis study provides an analysis of L-index thresholds for seven immunoturbidimetric assays. Due to the variety of human lipoproteins, limits defined using endogenously lipemic patient specimens may be different from those derived from spiking studies using Intralipid.

Engineering of anti-human interleukin-4 receptor alpha antibodies with potent antagonistic activity

Development of antagonistic antibody (Ab) against interleukin-4 receptor alpha (IL-4Rα) subunit of IL-4/IL-13 receptors is a promising therapeutic strategy for T helper 2 (TH2)-mediated allergic diseases such as asthma and atopic dermatitis. Here we isolated anti-human IL-4Rα antagonistic Abs from a large yeast surface-displayed human Ab library and further engineered their complementarity-determining regions to improve the affinity using yeast display technology, finally generating a candidate Ab, 4R34.1.19. When reformatted as human IgG1 form, 4R34.1.19 specifically bound to IL-4Rα with a high affinity (KD ≈ 178 pM) and effectively blocked IL-4- and IL-13-dependent signaling in a reporter cell system at a comparable level to that of the clinically approved anti-IL-4Rα dupilumab Ab analogue. Epitope mapping by alanine scanning mutagenesis revealed that 4R34.1.19 mainly bound to IL-4 binding sites on IL-4Rα with different epitopes from those of dupilumab analogue. Further, 4R34.1.19 efficiently inhibited IL-4-dependent proliferation of T cells among human peripheral blood mononuclear cells and suppressed the differentiation of naïve CD4+ T cells from healthy donors and asthmatic patients into TH2 cells, the activities of which were comparable to those of dupilumab analogue. Our work demonstrates that both affinity and epitope are critical factors for the efficacy of anti-IL-4Rα antagonistic Abs.

Phase II study to evaluate safety and efficacy of MEDI4736 (durvalumab) + radiotherapy in patients with newly diagnosed unmethylated MGMT glioblastoma (new unmeth GBM).

2032 Background: Durvalumab (durva), a human IgG1 monoclonal Ab against PD-L1, is FDA-approved for selected patients with bladder and non-small cell lung cancers. PD-L1 is expressed by some GBM tumors, while GBM infiltrating T lymphocytes often express PD-1. Radiation induced cell death releases tumor antigens and could potentiate anti-PD-(L)1 therapy. Methods: This ongoing Phase 2 open-label study (NCT02336165) evaluates the safety and efficacy of durva (10 mg/kg every 2 weeks) in 5 GBM cohorts. Results are presented for Cohort A, which evaluates durva + standard radiotherapy (RT, 60 Gy over 30 fractions) followed by durva monotherapy in patients with new unmeth GBM after maximum safe resection. The primary efficacy endpoint for Cohort A is overall survival at 12 months (OS12); secondary endpoints include safety/tolerability, tumor response rate, and progression-free survival (PFS). Historical benchmarks of median OS and OS12 for patients with new unmeth GBM following standard therapy are 12.7 months and 50%, respectively (EORTC 26981-22981/NCIC CE.3). Results: Median follow-up of 40 enrolled patients is 24.5 months (data cutoff = 05 Nov 2018). Baseline characteristics: male, 70%; median age, 57.0 [22 to 77] years; ECOG PS0, 60.0%; ECOG PS1, 40.0%; measurable disease, 80.0%; and dexamethasone use, 32.5%. Treatment-related adverse events with maximum CTCAE grade ≥ 3 occurred in 14 (35.0%) patients; the most common were asymptomatic increased lipase (n = 6) and increased amylase (n = 2). Twenty-four of 40 patients were alive at 12 months (Kaplan-Meier for OS12, 60.0% [90% CI: 46.1, 71.4]). Median OS was 15.1 (95% CI: 12.0, 18.4) months. As of 05 Nov 2018, 8 (20%) patients remain alive, with ongoing survival ranging from 15.7 to 34.9 months. Tumor immunocorrelative and systemic studies are pending. Conclusions: This is the first study report of anti-PD-L1 for new GBM. Durva was well tolerated when combined with RT and seemed to have efficacy among patients with new unmeth GBM. Further studies may be warranted. Clinical trial information: NCT02336165.

Evaluation of human platelet lysate (HPL) as a substitute for foetal bovine serum (FBS) and recombinant proteins for the growth and maintenance of bone marrow derived mesenchymal stromal cells (BM-MSC)

Aim To evaluate the potential of using HPL produced in Australia as a substitute for FBS during in vitro culture of BM-MSC. Background The replacement of FBS with an alternate serum source during the manufacture of cellular products for human therapeutics should minimise cost and exposure to the potential risk of animal products. Human AB sera serves as the current gold standard in clinical grade manufacture of cell therapies but suffers from high cost and limited supply. Here, we propose the use of HPL as an FBS replacement. Methods We compared BM-MSC cultured in FBS or HPL by assessing their growth, viability and phenotype by flow cytometry at P3 and P4. Three batches of HPL made from either apheresis or pooled platelet concentrates after expiry were tested. BM-MSC from a single donor were seeded at 1.8 × 103/cm2 in culture flasks pre-coated with 0.0001% human fibronectin, and passaged at 80% confluency. Cells were cultured in DMEM supplemented with 5% apheresis HPL, 10% pooled HPL or 10% Commercial HPL, compared to the Control media (10% FBS, 5 ng/mL PDGF, and 5 ng/mL EGF). As preliminary analysis revealed minimal variability between batches of HPL, we focused the remaining experiments on one batch of each type of HPL. Results The phenotype of all BM-MSC at P3 and P4 satisfied established MSC criteria, that is, >98% expression of CD73, CD90 and CD105, and lack of expression (i.e. 0.05 by one way ANOVA). In contrast, pooled HPL did not support BM-MSC growth beyond P3. This may be due to differences in the characteristics of apheresis and pooled HPL. Conclusion The growth and phenotype of BM-MSC cultured in either apheresis HPL or commercial HPL were comparable, suggesting apheresis HPL may prove a suitable alternative to FBS. A simpler preparation protocol of HPL for the end users will increase the likelihood of introducing HPL into current laboratory practice. The potential of pooled HPL warrants further investigation.

Results of point-of-care manufacturing of bispecific chimeric antigen receptor (CAR) LV20.19 CAR-TCELLS in a phase i study for relapsed/refractory (R/R), non-hodgkin lymphoma (NHL)

Background & Aim Current approved CAR-T cell manufacturing employs off-site production, a labor-intensive process that requires several weeks and involves shipping back and forth to the production site. This production model may limit availability and accessibility to CAR-T cells and delay treatment for patient (pt) s with rapidly progressive disease. To address these limitations, we utilized the Miltenyi CliniMACS Prodigy, a GMP-compliant device, to produce CAR-T cells on-site for a first-in-human bi-specific CD19/20 (LV20.19) CAR-T Phase I trial. Methods, Results & Conclusion CAR-T cells were manufactured within our Cell Therapy Laboratory, an ISO7 facility. Pt's mononuclear cell apheresis was collected and loaded onto the Prodigy to isolate CD4/CD8 T cells. The T cells were suspended in TexMACS supplemented with human AB serum and IL-2, activated with TransACT, and then transduced by LV20.19 the next day. Culture wash and feedings were done during the production process, and the final cell product was harvested at day 14. The process was automated with limited machine/operator interactions. CAR-T expression was assessed by protein L staining. CAR-T cells were either administered fresh or cryopreserved for later infusion. Dose was escalated in a 3+3 manner from 2.5e+5 cells/kg to a target dose of 2.5e+6 cells/kg. CAR-T cells have been successfully generated for 11/11 enrolled pts thus far with no manufacture failures, and all products met release criteria. Median T cell recovery from the apheresis products was 59.5% with a median CD3+ purity of 97.2% (Figure 1). Final product CAR expression was at a median of 17.6%. A median of 4.7e+8 CAR T cells was obtained at harvest, exceeding the required target dose. CAR-T cells contained both CD4 and CD8 T cells with higher CD4 content. The majority of CAR-T cells showed an effector-memory phenotype. CAR-T cells were able to kill CD19+/20+ target cells and produced IFN-gin response to antigens. Among the 11 treated pts 8 received fresh CAR-T cells and 3 received CAR-T cells after cryopreservation. As no DLTs were observed at the 3 dose levels, a dose of 2.5e+6 cells/kg has been selected for further expansion. Clinical response shows 6 pts have achieved a CR at Day +28 with a duration of response between 2 to 14 months. In summary, initial results demonstrated feasibility for point-of-care manufacturing of bispecific LV20.19CAR-T cells with safe and promising efficacy in treating heavily pretreated pts with R/R B-cell NHL, which warrants further investigations.

Monocytes subsets and cytokine levels in chronic Chagas disease

Abstract Monocytes are classified according to CD14 and CD16 expression into classical, intermediate and non classical. Variations in monocytes subsets are involved in the pathogenesis of protozoan and helminthic infections. This study assessed the relationship of monocyte subsets percentages and the level of cytokines in blood from Colombian chagasic patients. Donors included asymptomatic or indeterminate (IND), symptomatic or chronic cardiac chagasic (CCC) and heart transplant (HTCC) patients; also, non-chagasic cardiopathy patients (NCC) and healthy individuals (HI) as controls. Peripheral blood mononuclear cells (PBMCs) were isolated. Fc receptors were blocked using anti-CD16/CD32 and human AB serum. Cells were labeled with antibodies for CD14, CD16 and HLA-DR, and acquired in a flow cytometer. Cytokines were measured in plasma with a Human M1/M2 Macrophage kit. Comparisons were done with Kruskal-Wallis followed by Dunn’s post hoc test and correlations were evaluated by Spearman’s coefficient. Percentages of CD14+ CD16+ and CD14+ HLA-DR+ monocytes were higher in patients with heart involvement (CCC, HTCCC, and NCC). Percentage of intermediate monocytes (inflammatory) increased in CCC patients and was positively correlated with IL-6 concentration and negatively correlated with IL-12p40 levels. IND donors had higher percentages of classical monocytes and an increased production of CCL17, and they positively correlate. The results suggest that variations in the monocyte subsets and cytokines levels might be associated with protection (classical monocytes and CCL17) or the development (intermediate monocytes and IL-6) of chagasic myocardiopathy

Threshold for Pre-existing Antibody Levels Limiting Transduction Efficiency of Systemic rAAV9 Gene Delivery: Relevance for Translation

Widespread anti-AAV antibodies (Abs) in humans pose a critical challenge for the translation of AAV gene therapies, limiting patient eligibility. In this study, non-human primates (NHPs) with pre-existing αAAV Abs were used to investigate the impact of αAAV9 Ab levels on the transduction efficiency of rAAV9 via systemic delivery. No significant differences were observed in vector genome (vg) biodistribution in animals with ≤1:400 total serum αAAV9-IgG compared to αAAV9-Ab-negative animals, following an intravenous (i.v.) rAAV9-hNAGLUop (codon-optimized human α-N-acetylglucosaminidase coding sequence cDNA) injection. Serum αAAV9-IgG at >1:400 resulted in a >200-fold decrease in vg in the liver, but had no significant effect on vg levels in brain and most of the peripheral tissues. Although tissue NAGLU activities declined significantly, they remained above endogenous levels. Notably, there were higher vg copies but lower NAGLU activity in the spleen in NHPs with >1:400 αAAV9 Abs than in those with ≤1:400 Abs. We demonstrate here the presence of a threshold of pre-existing αAAV9 Abs for diminishing the transduction of i.v.-delivered AAV vectors, supporting the expansion of patient eligibility for systemic rAAV treatments. Our data also indicate that high pre-existing αAAV9 Abs may promote phagocytosis and that phagocytized vectors are not processed for transgene expression, suggesting that effectively suppressing innate immunity may have positive impacts on transduction efficiency in individuals with high Ab titers.